Mitochondrial DNA drives abscopal responses to radiation that are inhibited by autophagy.

Autophagy supports both cellular and organismal homeostasis. However, whether autophagy should be inhibited or activated for cancer therapy remains unclear. Deletion of essential autophagy genes increased the sensitivity of mouse mammary carcinoma cells to radiation therapy in vitro and in vivo (in immunocompetent syngeneic hosts). Autophagy-deficient cells secreted increased amounts of type I interferon (IFN), which could be limited by CGAS or STING knockdown, mitochondrial DNA depletion or mitochondrial outer membrane permeabilization blockage via BCL2 overexpression or BAX deletion. In vivo, irradiated autophagy-incompetent mammary tumors elicited robust immunity, leading to improved control of distant nonirradiated lesions via systemic type I IFN signaling. Finally, a genetic signature of autophagy had negative prognostic value in patients with breast cancer, inversely correlating with mitochondrial abundance, type I IFN signaling and effector immunity. As clinically useful autophagy inhibitors are elusive, our findings suggest that mitochondrial outer membrane permeabilization may represent a valid target for boosting radiation therapy immunogenicity in patients with breast cancer.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Cellular senescence in the response of HR+ breast cancer to radiotherapy and CDK4/6 inhibitors.

BACKGROUND: Preclinical evidence from us and others demonstrates that the anticancer effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors can be enhanced with focal radiation therapy (RT), but only when RT is delivered prior to (rather than after) CDK4/6 inhibition. Depending on tumor model, cellular senescence (an irreversible proliferative arrest that is associated with the secretion of numerous bioactive factors) has been attributed beneficial or detrimental effects on response to treatment. As both RT and CDK4/6 inhibitors elicit cellular senescence, we hypothesized that a differential accumulation of senescent cells in the tumor microenvironment could explain such an observation, i.e., the inferiority of CDK4/6 inhibition with palbociclib (P) followed by RT (P→RT) as compared to RT followed by palbociclib (RT→P). METHODS: The impact of cellular senescence on the interaction between RT and P was assessed by harnessing female INK-ATTAC mice, which express a dimerizable form of caspase 8 (CASP8) under the promoter of cyclin dependent kinase inhibitor 2A (Cdkn2a, coding for p16Ink4), as host for endogenous mammary tumors induced by the subcutaneous implantation of medroxyprogesterone acetate (MPA, M) pellets combined with the subsequent oral administration of 7,12-dimethylbenz[a]anthracene (DMBA, D). This endogenous mouse model of HR+ mammary carcinogenesis recapitulates key immunobiological aspects of human HR+ breast cancer. Mice bearing M/D-driven tumors were allocated to RT, P or their combination in the optional presence of the CASP8 dimerizer AP20187, and monitored for tumor growth, progression-free survival and overall survival. In parallel, induction of senescence in vitro, in cultured human mammary hormone receptor (HR)+ adenocarcinoma MCF7 cells, triple negative breast carcinoma MDA-MB-231 cells and mouse HR+ mammary carcinoma TS/A cells treated with RT, P or their combination, was determined by colorimetric assessment of senescence-associated ß-galactosidase activity after 3 or 7 days of treatment. RESULTS: In vivo depletion of p16Ink4-expressing (senescent) cells ameliorated the efficacy of P→RT (but not that of RT→P) in the M/D-driven model of HR+ mammary carcinogenesis. Accordingly, P→RT induced higher levels of cellular senescence than R→TP in cultured human and mouse breast cancer cell lines. CONCLUSIONS: Pending validation in other experimental systems, these findings suggest that a program of cellular senescence in malignant cells may explain (at least partially) the inferiority of P→RT versus RT→P in preclinical models of HR+ breast cancer.

Anticancer effects against colorectal cancer models of chloro(triethylphosphine)gold(I) encapsulated in PLGA-PEG nanoparticles.

Chloro(triethylphosphine)gold(I), (Et3PAuCl hereafter), is an Auranofin (AF)-related compound showing very similar biological and pharmacological properties. Like AF, Et3PAuCl exhibits potent antiproliferative properties in vitro toward a variety of cancer cell lines and is a promising anticancer drug candidate. We wondered whether Et3PAuCl encapsulation might lead to an improved pharmacological profile also considering the likely reduction of unwanted side-reactions that are responsible for adverse effects and for drug inactivation. Et3PAuCl was encapsulated in biocompatible PLGA-PEG nanoparticles (NPs) and the new formulation evaluated in colorectal HCT-116 cancer cells in comparison to the free gold complex. Notably, encapsulated Et3PAuCl (nano-Et3PAuCl hereafter) mostly retains the cellular properties of the free gold complex and elicits even greater cytotoxic effects in colorectal cancer (CRC) cells, mediated by apoptosis and autophagy. Moreover, a remarkable inhibition of two crucial signaling pathways, i.e. ERK and AKT, by nano-Et3PAuCl, was clearly documented. The implications of these findings are discussed.

The combined activation of KCa3.1 and inhibition of Kv11.1/hERG1 currents contribute to overcome Cisplatin resistance in colorectal cancer cells.

BACKGROUND: Platinum-based drugs such as Cisplatin are commonly employed for cancer treatment. Despite an initial therapeutic response, Cisplatin treatment often results in the development of chemoresistance. To identify novel approaches to overcome Cisplatin resistance, we tested Cisplatin in combination with K+ channel modulators on colorectal cancer (CRC) cells. METHODS: The functional expression of Ca2+-activated (KCa3.1, also known as KCNN4) and voltage-dependent (Kv11.1, also known as KCNH2 or hERG1) K+ channels was determined in two CRC cell lines (HCT-116 and HCT-8) by molecular and electrophysiological techniques. Cisplatin and several K+ channel modulators were tested in vitro for their action on K+ currents, cell vitality, apoptosis, cell cycle, proliferation, intracellular signalling and Platinum uptake. These effects were also analysed in a mouse model mimicking Cisplatin resistance. RESULTS: Cisplatin-resistant CRC cells expressed higher levels of KCa3.1 and Kv11.1 channels, compared with Cisplatin-sensitive CRC cells. In resistant cells, KCa3.1 activators (SKA-31) and Kv11.1 inhibitors (E4031) had a synergistic action with Cisplatin in triggering apoptosis and inhibiting proliferation. The effect was maximal when KCa3.1 activation and Kv11.1 inhibition were combined. In fact, similar results were produced by Riluzole, which is able to both activate KCa3.1 and inhibit Kv11.1. Cisplatin uptake into resistant cells depended on KCa3.1 channel activity, as it was potentiated by KCa3.1 activators. Kv11.1 blockade led to increased KCa3.1 expression and thereby stimulated Cisplatin uptake. Finally, the combined administration of a KCa3.1 activator and a Kv11.1 inhibitor also overcame Cisplatin resistance in vivo. CONCLUSIONS: As Riluzole, an activator of KCa3.1 and inhibitor of Kv11.1 channels, is in clinical use, our results suggest that this compound may be useful in the clinic to improve Cisplatin efficacy and overcome Cisplatin resistance in CRC.

New gold carbene complexes as candidate anticancer agents.

Three structurally related gold(I) carbene complexes with bulky hydrophobic ligands i.e. 1-3 were investigated in solution for further consideration as candidate anticancer agents. Cytotoxic assays were subsequently conducted on bone marrow-derived preosteoclast cell line of human origin (FLG 29.1) and human colon cancer cells (HCT-116). A far greater cytotoxic activity was measured for compound 1 against HCT-116 cells compared to 2 and 3; conversely, all compounds were highly and similarly active against FLG 29.1 cells. Results obtained for the reaction of complexes 1 and 2 with RNase A documented the occurrence of a weak interaction with this model protein and the formation of a tiny amount of the corresponding adduct. Moreover, a certain reactivity of the complex 2 was also detected toward GSH. The general implications of the obtained results are discussed.

Treatment with 8-OH-modified adenine (TLR7 ligand)-allergen conjugates decreases T helper type 2-oriented murine airway inflammation.

A strategy to improve allergen-specific immunotherapy is to employ new adjuvants stably linked to allergens. The study is addressed to evaluate the in vivo and in vitro effects of allergens [natural Dermatophagoides pteronyssinus 2 (nDer p 2) and ovalbumin (OVA)] chemically bound to an 8-OH-modified adenine. Humoral and cellular responses were analysed in allergen-sensitized and challenged mice by using conjugates (Conj) in a therapeutic setting. The in vitro activity of the conjugates on cytokine production induced by bone marrow dendritic cells and the co-culture system was also investigated. The nDer p 2-Conj treatment in nDer p 2-primed and challenged BALB/c mice reduced the numbers of eosinophils in bronchoalveolar lavage fluid and lung, airway allergen-driven interleukin-13 (IL-13) production in lung mononuclear cells and IgE, in comparison with nDer p 2-treated mice. The increase of IgG2a paralleled that of interferon-γ (IFN-γ) and IL-10 in allergen-stimulated spleen cells. Similar effects were elicited by treatment with OVA-Conj in an OVA-driven BALB/c model. The nDer p 2-Conj or OVA-Conj redirected memory T helper type 2 cells towards the production of IL-10 and IFN-γ also in C57BL/6 mice and when subcutaneously administered. Interleukin-10, IL-12 and IL-27 were produced in vitro by Conj-stimulated bone marrow dendritic cells, whereas IL-10 and IFN-γ were up-regulated in co-cultures of CD11c(+) and CD4(+) T cells from Conj-treated mice stimulated with allergen. Cytofluorometric analysis indicated that the Conj expanded IFN-γ- and IL-10- producing memory T cells. The Conj effects on IL-10(-/-) and IL-12(-/-) mice confirmed the role of IL-10 and IFN-γ in inducing a protective and balanced redirection the T helper type 2-mediated airway inflammation.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND: Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE: We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS: Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS: nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS: The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.

Assays and strategies for immunogenicity assessment of biological agents.

Some patients with chronic inflammatory diseases either do not respond to or lose their initial responsiveness to Tumor Necrosis Factor (TNF) inhibitor therapy. In these patients, the clinical response after switching to another anti-TNF drug suggests that lack of response is not related to the therapeutic target itself but immunogenicity. All biologics are potentially immunogenic and can induce the development of antidrug antibodies (ADAs). ADA formation is associated with lower serum drug levels, infusion reactions, and loss of response. Analytical methods for ADA detection include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance, and electrochemiluminescence. Currently, RIA and ELISA are the preferred methods due to a combination of reproducibility, sensitivity, and cost but have some limitations. There is no single available assay that has all pros and no cons, and therefore the use of more methods for the assessment of samples is a high priority.

Exploiting Tertiary Lymphoid Structures to Stimulate Antitumor Immunity and Improve Immunotherapy Efficacy.

Tumor-associated tertiary lymphoid structures (TLS) have been associated with favorable clinical outcomes and response to immune checkpoint inhibitors in many cancer types, including non-small cell lung cancer. Although the detailed cellular and molecular mechanisms underlying these clinical associations have not been fully elucidated, growing preclinical and clinical studies are helping to elucidate the mechanisms at the basis of TLS formation, composition, and regulation of immune responses. However, a major challenge remains how to exploit TLS to enhance naïve and treatment-mediated antitumor immune responses. Here, we discuss the current understanding of tumor-associated TLS, preclinical models that can be used to study them, and potential therapeutic interventions to boost TLS formation, with a particular focus on lung cancer research.

Unconventional strategy could be the future: From target to KRAS broad range treatment.

The RAS gene family comprises genes that regulate cell growth and differentiation. KRAS, a member of this family, is often mutated in different cancers, resulting in uncontrolled cell growth and tumor development. Recent clinical trial results on KRAS inhibition in NSCLC have defined the presence of a significant proportion of patients resistant to direct G12C inhibition. The presence of co-mutations and the occurrence of secondary resistance phenomena observed in preclinical and clinical settings partly justify these poor results. In addition, all other non-G12C mutations currently remain without specific strategies. Evidence of interactions between KRAS signaling and the TME suggests potential in vitro efficacy of immune checkpoint inhibitors. In this short paper, we have reviewed the most relevant data from recent conferences, with a focus on KRAS inhibitors resistance mechanisms and interactions with the peri-tumor immune system. Commentary.

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.

Quantification of beta-galactosidase activity as a marker of radiation-driven cellular senescence.

Cellular senescence is a permanent state of cell cycle arrest that can be triggered by different stressors, including cancer treatments (the so-called "therapy-induced senescence"), such as radiation therapy (RT). Although senescent cells do not proliferate, they remain metabolically active and play a critical role in tumor progression, metastasis, and response to therapy. Therefore, investigating the induction of cellular senescence upon RT treatment is a critical read out for investigating RT efficacy or combinatorial strategies in cancer research. Senescent cells are characterized by a plethora of markers, including an increased content and activity of lysosomes, which can be detected by the activity of the lysosomal enzyme senescence-associated ß-galactosidase. In this chapter, we present a protocol for the gold standard cytochemical method for quantification of the activity of the senescence-associated ß-galactosidase in irradiated murine breast cancer cells in vitro.

Between psychopathy and deviant socialization: A close look at the mafia men.

The Italian mafia organizations represent a subculture with values, beliefs and goals that are antithetical to and undermining of the predominant society. The conduct of individual members includes such extreme violence for material gain, it may at least superficially suggest a severe personality disorder. Since the first edition of the DSM and into the 21st century, various terms have been used, sometimes interchangeably, but over time inconsistently, to designate the mentality and practices of mafia members. Only recently has the psychology of mafia members become a focus of serious scientific study. Following broader national multicenter research, the present study aimed at investigating the possible differences in psychopathy between those mafia associates who had been convicted only of mafia association (Group A, bosses), and those who were also convicted of violent crimes (Group B, soldiers). The Psychopathy Checklist-Revised (PCL-R) was administered to n = 48 male inmates convicted of mafia association (Mage 45.0 years, SD 10.9, range 20-80 years); Group A consisted of n = 26 (54%) subjects, Group B n = 22 (46%). Most of the sample (73%) did not manifest psychopathy (PCL-R ≥ 25) nor Mann-Whitney U test disclosed significant differences in the total PCL-R scores between the study groups. We found significantly higher scores of PCR-R factor 1 (interpersonal / affective) in the members of the mafia association also convicted of violent crimes (PCL-R F1, group A: 5.8 ± 3.7; group B: 7.9 ± 3.5; p < 0.05), this difference appeared explainable on the basis of a higher component of affective psychopathy. These initial results add to the limited literature on mafia and psychopathy and seem to suggest the existence of a specific component of psychopathy in the subgroup of mafiosi with overtly violent conduct.

Challenges in the treatment of small cell lung cancer in the era of immunotherapy and molecular classification.

For many years the standard of care for small cell lung cancer (SCLC) has remained unchanged. Despite decades of active research, current treatment options are limited and the prognosis of patients with extended disease (ED) SCLC remains poor. The introduction of immune checkpoint inhibitors (ICIs) represents an exception and the only recent approval for ED-SCLC. However, the magnitude of benefit obtained with immunotherapy in SCLC is much more modest than that observed in other malignancies. Different pro-immunogenic or immunosuppressive features within the tumor microenvironment of SCLC may either modulate the sensitivity to immunotherapy or conversely dampen the efficacy of ICIs. Beside immunotherapy, a deeper understanding of the molecular biology of SCLC has led to the identification of new therapeutic targets for this lethal malignancy. Recent epigenetic and gene expression studies have resulted into a new molecular classification of four distinct subtypes of SCLC, defined by the relative expression of key transcription regulators and each characterized by specific therapeutic vulnerabilities. This review discusses the rationale for immunotherapy in SCLC and summarizes the main ICIs-trials in this tumor. We provide also an overview of new potential therapeutic opportunities and their integration with the new molecular classification of SCLC.

Early changes in circulating tumor DNA (ctDNA) predict treatment response in metastatic KRAS-mutated colorectal cancer (mCRC) patients.

The detection of RAS mutations and co-mutations in liquid biopsy offers a novel paradigm for the dynamic management of metastatic colorectal cancer (mCRC) patients. Expanding the results of the prospective OMITERC (OMIcs application from solid to liquid biopsy for a personalized ThERapy of Cancer) project, we collected blood samples at specific time points from patients who received a first-line chemotherapy (CT) for KRAS-mutated mCRC. CTC quantification was performed by CellSearch® system. Libraries from cfDNA were prepared using the Oncomine™ Colon cfDNA Assay to detect tumour-derived DNA in cfDNA. The analysis involved >240 hotspots in 14 genes. Twenty patients with KRAS-mutated mCRC treated at the Medical Oncology Unit of Careggi University Hospital were prospectively enrolled. Nine patients had available data for longitudinal monitoring of cfDNA. After 6 weeks of first-line CT an increase of KRAS-mutated clone was reported in the only patient who did not obtain disease control, while all patients with decrease of KRAS clones obtained disease control. Overall, in patients with a short (<9 months) progression-free survival (PFS) we registered, at 6 weeks, an increase in cfDNA levels and in KRAS mutations or other co-mutations, i.e. PIK3CA, FBXW7, GNAS, and TP53. In selected cases, co-mutations were able to better anticipate radiological progressive disease (PD) than the increase of KRAS-mutated clones. In conclusion, our study confirms plasma ctDNA as a crucial tool for anticipating PD at an early time point and highlights the value of a comprehensive assessment of clonal dynamics to improve the management of patients with mCRC.

Drug-specific Th2 cells and IgE antibodies in a patient with anaphylaxis to rituximab.

Rituximab (RTX) is currently used in the treatment of lymphoproliferative diseases and of several rheumatologic disorders and is a frequent cause of acute infusion reactions, usually classified as cytokine release syndrome (CRS). Some infusion reactions to RTX raise concern for immediate type I hypersensitivity, even if to date RTX-specific IgE antibodies have not been reported. To improve knowledge of the mechanisms of reactions to RTX, we investigated humoral and cellular immune responses to this drug in a patient suffering from rheumatoid arthritis who displayed two immediate infusion-related reactions. RTX-exposed tolerant patients and healthy untreated subjects were used as controls. Non-isotype-specific and IgE anti-RTX antibodies were positive in the serum samples collected from the reactive patient but not in those from the control groups. Only the reactive patient also displayed skin testing positivity with RTX. More importantly, RTX-stimulated peripheral blood mononuclear cells from the reactive patient, but not from the controls, displayed a dose-dependent proliferative response associated with a Th2 cytokine production profile. Our results show the presence of RTX-specific Th2-type cells and IgE antibodies, thus suggesting that type I hypersensitivity may be an additional mechanism to CRS in the development of RTX reactions.

Targeting oncogene and non-oncogene addiction to inflame the tumour microenvironment.

Immune checkpoint inhibitors (ICIs) have revolutionized the clinical management of multiple tumours. However, only a few patients respond to ICIs, which has generated considerable interest in the identification of resistance mechanisms. One such mechanism reflects the ability of various oncogenic pathways, as well as stress response pathways required for the survival of transformed cells (a situation commonly referred to as 'non-oncogene addiction'), to support tumour progression not only by providing malignant cells with survival and/or proliferation advantages, but also by establishing immunologically 'cold' tumour microenvironments (TMEs). Thus, both oncogene and non-oncogene addiction stand out as promising targets to robustly inflame the TME and potentially enable superior responses to ICIs.

A Rapid Fatal Outcome of Iliopsoas Hematoma: Clinical and Autopsy Findings.

A case of massive muscular bleeding of iliopsoas resulting in lethal exsanguination is presented. The intramuscular bleeding occurred spontaneously in an old man with heart failure, presented to the emergency department after the acute onset of shortness of breath, and treated with therapeutic doses of antiplatelets and heparin to prevent thrombosis. On the sixth day of recovery, pain in the left lumbar region develops while there was a decrease in hemoglobin level. Computed tomography (CT) scan revealed a 10 × 3 cm hematoma of the left iliac muscle. The treatment was immediately stopped, but within 6 hours, the death was confirmed. The autopsy revealed that the hematoma, and its increased size since the latest imaging assessment, was the leading cause of death. Particularly in older patients with comorbidity, even in those with clotting parameters in the therapeutic range, the potential for fatal result of iliopsoas muscle bleeding should be considered. Identifying potential patience with increased risk of this complication could be important, especially in pandemic time of COVID-19, when the use of anticoagulant therapy-both for treatment and for prevention of severe disease-has become massive and addressed also to people without previous and specific pathologies.

Radiotherapy as a tool to elicit clinically actionable signalling pathways in cancer.

A variety of targeted anticancer agents have been successfully introduced into clinical practice, largely reflecting their ability to inhibit specific molecular alterations that are required for disease progression. However, not all malignant cells rely on such alterations to survive, proliferate, disseminate and/or evade anticancer immunity, implying that many tumours are intrinsically resistant to targeted therapies. Radiotherapy is well known for its ability to activate cytotoxic signalling pathways that ultimately promote the death of cancer cells, as well as numerous cytoprotective mechanisms that are elicited by cellular damage. Importantly, many cytoprotective mechanisms elicited by radiotherapy can be abrogated by targeted anticancer agents, suggesting that radiotherapy could be harnessed to enhance the clinical efficacy of these drugs. In this Review, we discuss preclinical and clinical data that introduce radiotherapy as a tool to elicit or amplify clinically actionable signalling pathways in patients with cancer.